After two decades of intense research in HIV vaccine development, recent successes with several commercially viable vaccine candidates now offer hope. Both DNA, and DNA boosted with poxvirus, have induced robust T cell responses and conferred partial protection macaques. These achievements are modest, however, when compared to the obstacles that must be overcome to protect diverse human populations from HIV isolates that are considerably more hypervariable than the viruses used in these studies. SAVINE (scrambled antigen vaccine), a novel vaccine developed by Ian Ramshaw and Scott Thompson, addresses this concern. SAVINE vaccines are composed of randomly rearranged overlapping peptide fragments (30 amino acids overlapping by 15 amino acids) from all viral structural and regulatory genes using a systematic and synthetic approach in which the size and processing mechanisms of T cell epitopes are the primary criteria for determining how antigens might be disrupted. Importantly, no effort is made to bias the design towards known T cell epitopes. Thus, specific viral epitopes, and the predominant HLA haplotype of the population at risk need not be known. In this application, we propose to evaluate the immunogenicity and efficacy of the SAVINE approach using the SIV macaque model for AIDS. SAVINE composition will be optimized by comparing the sequence and immunogenicity of a prototypic HIV SAVINE to those present in a standard clade B vaccine (aims 1 and 3). Using this information, SIV SAVINEs will be constructed by the founders of the SAVINE vaccine approach, Drs. Scott Thompson and Ian Ramshaw (aim 2). Once SIV SAVINE vaccines are proven immunogenic in mice (aim 2), they will be evaluated for their ability to induce both mucosal and systemic immune responses, and to confer mucosal protection, in macaques (aim 4). The ability of SAVINE DNA and SAVINE avipox, both alone and in combination, to induce mucosal, as well as systemic, immune responses will be determined. DNA vaccines will be further potentiated by co-delivery of the gene for E. coli enterotoxin, a combination that we have recently shown to significantly augment T cell responses induced by whole gene immunization in macaques. The breadth and magnitude of T cell responses will be evaluated in both periphery and gut associated tissues (jejunal lamina propria, mesenteric lymph nodes) so that the optimum strategy for inducing mucosal responses, and the relationship of these responses to protection, can be determined. Immune responses in both mice and monkeys will be evaluated by Powderject Vaccines so that these responses can be directly compared with each other, as well as to historical data with other DNA vaccines. Although other studies have shown that a combined DNA:avipox strategy induces better systemic immune responses than either vaccine alone, for the first time, the ability of the combined DNA: avipox strategy to induce mucosal immune responses will be directly compared to either approach alone. Because systemic responses are often poor predictors of mucosal responses, these studies should provide important new information in HIV vaccine development. Together, these experiments will provide data essential to the translation of these studies to human trials.